The immune responses to Pneumocystis are poorly understood, but cytokines may play a role in both clearing Pneumocystis infection and in the hypoxia associated with Pneumocystis pneumonia that may be exacerbated following initiation of therapy. We are using the scid mouse model, as well as other immunodeficient mice, to further evaluate the role of individual cytokines and other immunoregulatory molecules in modulating Pneumocystis infection. We have developed a real time PCR assay to quantitate Pneumocystis over a wide dynamic range, and are currently examining Pneumocystis infection in healthy animals to better understand immune responses in the normal host. Using microarrays, we have been able to identify genes that are differentially expressed during Pneumocystis infection in healthy compared to immunodeficient mice; the majority of differentially expressed genes are related to the immune system. Immunohistochemical studies of the lungs done in parallel have demonstrated that there is an influx of CD4 cells and macrophages at 5 weeks, and, surprisingly, an influx of B cells at 6 weeks in immunocompetent mice but not CD40L KO mice. Microarray studies suggest that the B cells persist through at least 10 weeks, well after the infection has been cleared. We have extended these studies to examine immune responses in other immunodeficient mice, including those with defective innate immune responses, such as Myd88 knockout mice which are deficient in TLR signals, and CDld KO mice, which lack NKT cells. Preliminary studies have shown that these mice are not susceptible to uncontrolled Pneumocystis infection, suggesting that neither TLN signaling, nor NKT cells are critical to controlling Pneumocystis infection. In collaboration with Philip Murphy and his group, we have examined changes in expression of a variety of chemokine and chemokine receptor genes, using Taqman real-time PCR assays, and have been able to identify CCR-2 and its ligands as potentially important in the response to Pneumocystis infection. We are thus in the process of examining the susceptibility of CCR-2 knock-out mice to Pneumocystis infection. To allow us to evaluate immune responses more easily, we have developed a multiplex assay that allows the evaluation of expression levels of 30 genes simultaneously, using branched DNA amplification combined with Luminex bead technology. We will use this assay to examine the lungs of various strains of knock-out mice at different stages of Pneumocystis infection, to determine if their immune responses are similar to those seen in healthy, immunocompetent mice. It is hoped that these studies will provide insights into the mechanisms critical for control of Pneumocystis infection in the healthy host, and may provide mechanisms for increasing clearance of Pneumocystis or decreasing the inflammation that may be causing hypoxia.